5
Figure 3. Mass chromatograms of leu-enkephalin (60 pg/mL). The top chromatogram corresponds to injection #5, and the bottom chromatogram to injection #111. There are no significant changes in peak area, tR
, and peak shape. Figure 2A
Comparison of standard calibration plots after prolonged use of an emitter would give an indication of the condition of the emitter. Initial and final calibration plots (after more than one hundred injections) for each of the three neuropeptides were compared (data not shown). Linearity did not change: if the emitter had clogged at any point over this continuous run, differences in the initial and final calibration plots would have been observed because of signal deterioration.
To further inspect emitter clogging, the physical appearance of emitters used for one month and unused emitters were examined under a light microscope. Photomicrographs did not reveal any clogging.
The flow rate used in this work (900 nL/min) is high for nano-scale work, yet the emitters tested did not show signs of deterioration due to clogging. Thus, using freshly produced ultrapure water delivered out of a 0.22 µm membrane filter at the water purification system’s point of use is strongly recommended for nano- LC/MS analyses.
Conclusions Figure 2B:
Mass spectra of bradykinin in fresh ultrapure water (2A) and mass spectra of bradykinin in ultrapure water spiked with sodium ions (2B)
It is important to keep organic contamination at a minimum since organic contaminants could accumulate in reversed-phase columns and eventually elute as extraneous peak(s) in the chromatogram. A dual-wavelength UV lamp (185 + 254 nm) in the water purification chain (Figure 1) oxidises organic contaminants,
reducing the levels to ≤ 5 ppb. At the end of the water purification chain, a 0.22 µm membrane point-of-use filter retains bacteria and particulates that could clog emitters. All these technologies work together to provide ultrapure water that is free of any contaminants that could potentially interfere with nano-LC/MS analyses.
Experiments were also carried out to verify that fresh ultrapure water does not clog emitters. Figure 3 shows mass chromatograms of leu-enkephalin where fast LC conditions were used10
for over one hundred back-to-back injections, with fresh
ultrapure water making up the aqueous mobile phase. Retention times and peak areas remained essentially unchanged between early (#5) and late (#111) injections. Comparisons of the mass chromatograms of met-enkephalin and angiotensin (not shown) also remained constant. The excellent run-to-run reproducibility indicates that there was no clogging of the emitter.
In another set of experiments, more than 500 injections of neuropeptides were performed over a month-long period using various nano-bore columns from different vendors placed in-line with a single MonoSpray emitter. At the end of the month, the initial column used in the experiment was placed back in-line to confirm the integrity of the emitter. The peak shapes and retention times observed were essentially the same as the ones obtained at the beginning of the experiment. These suggest that the emitter maintained its integrity after hundreds of injections using fresh ultrapure water as a component of the mobile phase.
The consistent quality and minimal levels of organic and ionic contaminants in fresh ultrapure water are crucial in robust and sensitive nano-LC/MS analyses of biological samples. This work is a proof of concept that nano-LC/MS eluent prepared using freshly produced ultrapure water filtered through a 0.22 µm point-of-use filter does not cause emitter clogging. Neuropeptide retention times, peak areas, and calibration plots were unchanged after extended use of the emitters. No evidence of any debris or clogging was shown in photomicrographs of a used emitter.
References 1. M. Noga, et. al., J. Sep. Sci. 30, 2179 – 2189 (2007). 2. T.D. Wood, et. al., App. Spec. Rev. 38, 187-244 (2003). 3. I. Manisali, D.D.Y. Chen, B.B. Schneider, Trends Anal. Chem. 25, 243-256 (2006). 4. B.A. Sinnaeve, J.F. Van Bocxlaer, J. Chrom. A 1058, 113-119 (2004). 5. H. Liu, et. al., J. Chrom. A 1147, 30-36 (2007).
6. A. El-Faramawy, K.W.M. Siu, B.A. Thomson, J. Am. Soc. Mass Spectrom. 16, 1702-1707 (2005).
7. R. Ramanathan, et. al., J. Am. Soc. Mass Spectrom. 18, 1891-1899 (2007). 8. J.M. Saz and M.L. Marina, J. Sep. Sci 31, 446-458 (2008).
9. M. Noga, F. Sucharski, P. Suder, and J. Silberring, J. Sep. Sci. 30, 2179-2189 (2007).
10. M.J. Hayward, M.D. Bacolod, Q.P. Han, M. Cajina, Z. Zou, Chapter 11 - Techniques to Facilitate the Performance of Mass Spectrometry: Sample Preparation, Liquid Chromatography, and Non-Mass-Spectrometric Detection in “Mass Spectrometry in Drug Metabolism,” M. Lee, M. Zhu, ed., Wiley, New York: 2010.
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